RU2672595C2 - Method for diagnosing molecular phenotype of patients suffering from diseases accompanied by chronic inflammation - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: proposed group of inventions relates to medicine. Proposed are a method and a set for diagnosing the molecular phenotype of patients suffering from a disease accompanied by chronic inflammation, and classified as "Th2-high", "Th2-low", "Th1-high" or "Th1-low". Proposed is a medication for the treatment of such patients, containing a DNA-enzyme specific for GATA-3 or Tbet.EFFECT: proposed group of inventions provides effective methods for determining the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation, and medications that are effective for the identified subgroup of patients.9 cl, 9 dwg, 1 tbl, 7 ex

Description

The invention relates to a method and kit of reagents for diagnosing the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation, as well as to a medicament for treating such a patient.

Chronic inflammation is an increasingly expanding range of medical problems of great socioeconomic importance. These include, in particular, the following groups of diseases: autoimmune diseases and diseases of a rheumatic clinical and morphological group (manifestations, for example, on the skin, lungs, kidneys, circulatory system, nervous system, connective tissue, musculoskeletal system, endocrine system ), immediate allergic reactions and asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, psoriasis and contact eczema, and chronic rejection reactions after organ and bone marrow transplantation. In recent decades, many of these diseases have become increasingly dominant not only in industrial regions, but partly around the world. So, already over 20% of the population in Europe, North America, Japan and Australia suffer from allergic diseases and asthma. Chronic obstructive pulmonary disease today ranks fifth among the most common causes of death worldwide, and by 2020, WHO estimates that it will take third place. The leading position in the statistics of morbidity and mortality worldwide belongs to atherosclerosis with its accompanying diseases - myocardial infarction, stroke and peripheral artery occlusion. Psoriasis and contact eczema, along with neurodermatitis, are the most common chronic inflammatory skin diseases.

In view of the still insufficiently studied interaction between environmental factors and a genetic predisposition, regulation of the immune system in the long term often happens incorrectly. Moreover, for such various diseases, the following general principles can be stated:

(A) The development of an excessive immune response to normally antigen-safe for humans. These antigens can be parts of the environment (for example, allergens, including pollen, animal hair, food, ticks, chemicals, including preservatives, dyes, cleaners and detergents). In such cases, patients develop allergic reactions. For example, active and passive smokers experience chronic obstructive pulmonary disease (COPD). On the other hand, the immune system can also respond to the components of its own organism, perceive them as foreign and initiate an inflammatory reaction against them. In such cases, an autoimmune disease develops. In each such case, the harmless, non-toxic antigens are mistakenly recognized as foreign or dangerous, and an inadequate inflammatory response is triggered.

(B) Diseases occur in several phases, which include initiation, progression, i.e. the development of an inflammatory reaction, and the associated destruction and rearrangement with the loss of the organ's functional capabilities (the so-called remodeling).

(C) Diseases exhibit patient-specific subphenotypic signs of manifestation.

(D) The components of innate and acquired immunity are continuously involved in the processes of initiation, maintenance, and also destruction and rearrangement. Under the influence of innate immunity (important components: antigen-presenting cells with their diverse populations and complement system), the activation and differentiation of cells of the adaptive immune system (important components: T and B lymphocytes) occurs. T cells in the further course assume central functions by differentiation into highly specialized effectors.

At the same time, they activate and acquire certain effector mechanisms, which, in particular, include the following functions: antibody formation, control of the functional capabilities of the effector cells of the immune system (for example, neutrophilic, basophilic, eosinophilic granulocytes), feedback from the functions of the innate immune system, effect on the functionality of non-hematopoietic cells, for example, epithelium, endothelium, connective tissue, bones and cartilage, and especially neuronal cells. There is a special interaction between the immune and nervous systems, on the basis of which the concept of neuro-immunological interaction was formed in chronic inflammation.

Since the already mentioned T-cells take on central functions during the course of the disease, an understanding of their specialization plays a crucial role. In the differentiation of naive (inactive) CD4 ⁺ cells into Th1 and / or Th2 cells, complex signaling cascades are involved.

Stimulation with the corresponding peptide HCH complex (the main histocompatibility complex) through the T-cell receptor causes clonal expansion and programmed differentiation of CD4 ⁺ T-lymphocytes into T-helper cells [Helper T cells] Th1 or Th2 cells. These two subtypes differ depending on their cytokine profiles. Th1 cells produce interferon-γ (INFγ), interleukin 2 (IL-2) and tumor necrosis factor-α, while Th2 cells secrete IL-4, IL-5, IL-9 and IL-13. Bacterial and viral infections elicit an immune response in which Th1 cells predominate. On the other hand, Th2 cells regulate the production of immunoglobulin Ε IgE antibodies against parasites. Moreover, between Th1 and Th2 cells there is an equilibrium. Violation of this balance leads to the occurrence of diseases, for example, an excessive Th1 cell response is associated with autoimmune diseases, while allergic diseases are based on a too strong Th2 cell response.

Th1 cytokines are known to be involved in the pathogenesis of autoimmune diseases, for example, autoimmune uveitis, experimental allergic encephalomyelitis, type 1 diabetes mellitus or regional enteritis (Crohn's disease), while Th2 cytokines (IL-4, IL-5, IL -13 and / or IL-9) are involved in the occurrence of chronic inflammatory diseases of the respiratory tract, for example, airway eosinophilia, asthma, mucus hypersecretion, and airway hyperresponsiveness. The basis of these diseases are pathophysiological changes during the formation of typical cytokines due to antigen-specific Th cells. Subpopulations of Th2 cells in the lung and respiratory tract cause characteristic symptoms of bronchial asthma in an experimental animal model.

In the occurrence of autoimmune diseases and chronic inflammatory reactions, two transcription factors are involved, among other things: TM-cell-specific transcription factor Tbet and Th2-cell-specific transcription factor GATA-3.

The Th1 cell-specific transcription factor Tbet is primarily responsible for the differentiation of naive CD ⁴ + T cells into Th1 cells. Its expression is controlled by the signal transduction pathways of the T cell receptor (TCR) and via the INFγ receptor / STAT1 (interferon-gamma receptor). Tbet transactivates the endogenous INFγ gene and causes the production of INFγ. Tbet function in vivo is also confirmed in knocked out mice (Tbet - / -). In Tbet-deficient mice, the number of Th2 cytokines is increased.

Tbet function is known in mucosal T cells in the event of inflammatory bowel disease. The transcription factor Tbet primarily causes the development of Th1 cells and controls the production of interferon-gamma INFγ in these cells. Due to inhibition of Tbet, the balance between Th1 and Th2 cells shifts toward Th2 cells.

Many inflammatory diseases, such as allergic asthma, in contrast, are associated with the activation of Th2 cells. Th2 cells perform an essential function in the development of allergic diseases, in particular, various asthmatic diseases. The required differentiation of Th0 cells into Th2 cells depends on the transcription factor GATA-3. GATA-3 is a member of the GATA family of transcription factors.

The Th2 cell-specific transcription factor GATA-3 is primarily responsible for the differentiation of naive CD4 ⁺ T cells into Th2 cells. In this case, the differentiation of Th2 cells is controlled mainly by two signal transmission paths: along the pathway of the T-cell receptor (TCR) and along the pathway of the IL-4 receptor. The signals transmitted from TCR activate both Th2 cell-specific transcription factors c-Maf and GATA-3, as well as transcription factors NFAT and AP-1. Activation of the IL-4 receptor leads to the binding of STAT6 to the cytoplasmic domain of the IL-4 receptor, where it is phospholated by Jak1 and Jak3 kinases. In turn, phosphorylation leads to dimerization and translocation of STAT6 into the cell nucleus, where STAT6 activates the transcription of GATA-3 and other genes. GATA-3 is a transcription factor with domains such as zinc fingers, which is expressed exclusively in mature Th2 cells, but not in Th1 cells.

Th2 cells produce cytokines, for example, IL-4, IL-5, IL-6, IL-13 and GM-CSF. Polarization in Th2 inhibits Th1 differentiation by inhibiting Tbet and vice versa. However, the expression of GATA-3 is not limited only to T cells. GATA-3 expression has been identified in eosinophilic and basophilic granulocytes, mast cells (mast cells) and epithelial cells. GATA-3 plays a central role in the immunopathogenesis of chronic inflammatory diseases, in particular allergic asthma.

Commonly recognized drugs for the treatment of chronic inflammatory diseases include, but are not limited to, corticosteroids, antileukotrienes (leukotriene receptor inhibitors), immunosuppressive drugs, and monoclonal antibodies with antimmunoglobulin E. However, the positive response of asthmatic patients to these therapeutic agents is manifested in different ways. For a long time, it was unclear what these differences in effectiveness are associated with. Ultimately, the therapy appropriate for the patient in each particular case had to be selected more or less by trial and error.

However, only recently it was possible to establish that, for example, patients with asthma can be divided into even smaller subgroups (Woodruff et al., 2009, hel-helper Type 2-driven Inflammation Defines Major Subphenotypes of Asthma (“T-helper 2- type-dependent inflammation determines the underlying subphenotypes of asthma ”), Am J Respir Crit Care Med, Vol 180, 388-395). So, it was shown that among patients with asthma there are at least two subgroups, designated as “Th2-high” (Th2 high) and “Th2-low” (Th2 low). Moreover, the “Th2-high” subgroup of patients with asthma is characterized by, among other things, increased expression of the POSTN gene encoding periostin for protein, as well as genes for IL-13 and IL-5. The "Th2-low" group of asthma patients tested showed low POSTN gene expression compared to the control group of healthy people. These different molecular phenotypes could be the cause of the different efficacy of common therapeutic agents. So, for the “Th2-high” subgroup, it was possible to identify an improved response to corticosteroid treatment.

In addition, it was found that two groups of patients with asthma, namely “Th2-high” and “Th2-low”, show a different response to therapy with a humanized monoclonal antibody against IL-13 (Corren et al., 2011, Lebrikizumab Treatment in Adults with Asthma (Treatment of Asthma in Adults), The new england journal of medicine, 10.1056 / NEJMoa1106469). Moreover, patients with asthma were first empirically assigned to the “Th2-high” group if the serum IgE values were higher than 100 IU / ml, and the number of eosinophilic granulocytes was 0.14 × 10 ⁹ cells per liter or more. Each patient whose indicators were below these values was assigned to the “Th2-low” group. As an alternative, classification was carried out depending on the level of periostin in the blood serum. This level serves as a surrogate marker for IL-13 th2 cytokines difficult to detect in the blood or respiratory tract. The fact that IL-13, among other things, causes the expression of the periostin-encoding POSTN gene in in vitro epithelial cells is used (Woodruff et al., 2007, Proc Natl Acad Sei USA., 104 (40): 15858-63. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids al., 2011, were assigned to the “periostin-high” group. As a rule, for these subgroups “Th2-you Cursed "and" high-periostin "was able to describe a more effective response to treatment with anti-IL-13- antibodies.

The authors of WO 2009/124090 A1 also proposed a certain classification of patients with asthma, and the expression of several candidate genes, for example, POSTN, CLCA1 and SERPINB2, is involved in this classification. Since the regulation of these genes by TP2 cytokines IL-4 or IL-13 is known to occur with increased expression, this cluster is also referred to as the "IL-4 / IL-13 gene expression profile" ("IL-4 / IL-13 -Signature "). In addition to determining the level of periostin in the blood serum, as well as the corresponding amount of mRNA (matrix, or messenger RNA), the determination of serum IgE values and the determination of the amount of eosinophilic granulocytes are described.

The drawback of stratifying patients based on this gene expression, primarily POSTN, is that along with the IL-4 / IL-13 gene expression profile, the IL-5 cytokine plays a significant role in asthma. In addition, the role of periostin protein in the cascade of immune responses, and therefore in the onset of the disease, is unknown.

Thus, the task is to find a biomarker that is suitable for reliable and at the same time convenient for clinical use determination of the molecular phenotype of patients suffering from any disease accompanied by chronic inflammation, assigning them to the groups “Th2- high ”or“ Th2-low ”and / or“ Th1-high ”or“ Th1-low ”. Further, the patient classified in this way should be treated with a therapeutic agent that is particularly effective for this particular subgroup. The biomarker should make it possible to individually predict the effectiveness of a therapeutic agent for a particular patient, in particular a patient with asthma.

According to the invention, this problem is solved by a method for diagnosing the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation by selecting the molecular phenotype from the group which includes the subgroups "Th2-high", "Th2-low", "Th1-high" and " Th1-low, ”and by quantifying the expression of the GATA-3 and / or Tbet genes in the patient’s biological isolate and its use to refer to any molecular phenotype of the disease. Moreover, a more accurate assignment to a specific type of patient suffering from a disease accompanied by chronic inflammation is carried out by quantitatively determining the expression of the genes for the transcription factor GATA-3 and / or transcription factor Tbet. As noted above in the introduction, the occurrence of autoimmune diseases and chronic inflammatory reactions involves Th1 cell-specific transcription factor Tbet and Th2 cell-specific transcription factor GATA-3. Polarization in Th2 inhibits differentiation into Th1 cells by inhibiting Tbet and vice versa. Depending on the level of expression of GATA-3 and / or Tbet, it is possible to attach to one or another molecular phenotype, i.e. attribution to a subgroup of a disease accompanied by chronic inflammation. The diagnostic method according to the invention allows with a high degree of information content to easily determine the molecular phenotype in everyday clinical conditions.

Transcription factors GATA-3 and Tbet are central key molecules in the occurrence of Th1 and / or Th2-dependent chronic inflammatory diseases. Therefore, direct quantification of protein expression and / or mRNA expression is the best approach to stratification of patients, since the result cannot be distorted by any intermediate mechanisms.

According to a preferred embodiment of the method, the expression level of GATA-3 and / or Tbet is determined by the amount of protein or mRNA. In this case, the amount of protein can be determined using immunoassay. An immunoassay is preferably an enzyme-linked immunosorbent assay ELISA (Enzyme-linked Immunosorbent Assay, ELISA assay), radioimmunoassay RIA (radioimmunassay, RIA), electrochemiluminescent ECL immunoassay (electrochemiluminescence immunoassay, ECL), enzyme-linked immunosorbent assay CLIA), solid-phase immunoassay with fluorescence amplification (fluorescence-linked Immunosorbent assay, FLIA) or multiplex immunoassay (multiplex-assay).

The advantage of all these types of analysis, along with high sensitivity and specificity, is the ability to automate, so they are all perfect for everyday use in a clinical setting. Of course, in the framework of the present invention, if necessary, any other suitable study can be selected to quantify the amount of GATA-3 and / or Tbet proteins. Further, the expression level of GATA-3 and / or Tbet can be determined by mass spectrometric methods, chromatographic methods, for example, using gas chromatography, liquid methods with separation of the solid phase, for example, using high performance liquid chromatography (HPLC), or methods based on micro- and nanofluidic technologies.

If necessary, to determine the expression of GATA-3 or Tbet using ELISA, the method may include the following steps:

- obtaining a lysate by cell lysis;

- adding a lysate to the well of a microtiter plate coated with a layer of the first antibody specific for GATA-3 or Tbet;

- washing microtiter tablet;

- adding a second specific for GATA-3 or Tbet antibodies in the well of microtiter plate;

- washing microtiter tablet;

- identification and quantification of GATA-3 or Tbet protein.

For detection, a second specific antibody can be labeled, for example, with biotin, and an enzyme associated with streptavidin can be separately added. However, the second specific antibody may be directly linked to the enzyme. If appropriate, it is also possible to use a third enzyme-linked antibody directed against the second specific antibody.

The enzyme, which is preferably peroxidase or alkaline phosphatase, is transferred by a suitable substrate suitable for colorimetry or chemiluminescence and the like.

According to another aspect of the present invention, in addition to or as an alternative to the method for protein counting, it is possible to determine the amount of GATA-3 and / or Tbet mRNA. PCR (polymerase chain reaction) is preferably suitable for this, particularly preferably quantitative PCR or microarray analysis. The selection procedure for GATA-3 and Tbet-specific probes and / or primers for these detection methods is known to a person skilled in the art.

The biological isolate according to a preferred embodiment of the method was obtained from whole blood, urine, sputum, swabs from bronchoalveolar lavage (BAL), biopsy, brush biopsy, cerebrospinal fluid, tracheal secretion, seminal fluid, hydroponic fluid, saliva, punctate or lymphatic fluid. Standard methods for preparing a suitable biological isolate are known to the skilled person.

GATA-3 and Tbet are proteins that exhibit their effect as transcription factors in the nucleus of T-helper cells of the Th1 and Th2 subtypes. To determine the concentration of both of these nuclear proteins in a certain volume of biological isolate, for example, in a certain volume of whole blood or the like, the cells forming GATA-3 and Tbet must first be isolated and then lysed. Direct detection of these proteins from human serum or plasma is hardly possible, because they are contained there in a concentration undetectable. Therefore, the analysis of GATA-3 and Tbet, if necessary, is carried out in four stages:

- separation and isolation of GATA-3 / Tbet-expressing cells from other cellular components of whole blood

- cell lysis and isolation of intracellular / nuclear proteins

- quantitative determination of the concentration of GATA-3 and Tbet and

- rationing of the detected concentrations of GATA-3 and Tbet.

According to a preferred refinement, the method according to the invention, regardless of whether the expression level of GATA-3 and / or Tbet is determined by the number of proteins or mRNA, also includes one or more of the following steps:

(i) isolation of white blood cells, preferably by centrifugation in a density gradient of ficoll;

(ii) increasing the concentration of leukocytes, preferably by gel filtration; or

(iii) increasing the concentration of Th1 / Th2 cells, in particular CD4 ⁺ T cells, using cell-specific antibodies, which are preferably associated with magnetic microcarrier beads.

The indicated steps (i) - (iii) are performed before cell lysis and, in each case, can increase the sensitivity as well as the information content of the diagnostic method, since GATA-3 and Tbet genes are expressed differently in leukocytes in different ways.

According to another aspect of the present invention, the assignment of a patient to a particular molecular phenotype of a “Th2-high” subgroup occurs when at least one of the following conditions is fulfilled:

a) expression of GATA-3 genes in a biological isolate above a predetermined control value,

b) a ratio of the expression levels of GATA-3 Tbet genes in the biological isolate above a predetermined control value.

Thus, the “Th2-high” subgroup is characterized by high absolute expression of GATA-3 genes compared to a given control value. At the same time, as a control value - in the case of determining the amount of proteins - the corresponding value of the GATA-3 protein content in the isolate of a healthy person can be used. However, absolute reference values are also possible. In the case of determining the amount of mRNA as a control value, the corresponding value of the amount of GATA-3 mRNA in the isolate of a healthy person can be used. However, again, absolute reference values are possible, for example, the number of copies / ml.

The assignment of a patient to the indicated molecular phenotype of a “Th2-high” subgroup in accordance with a preferred embodiment of the method according to the invention occurs if, as an alternative or in addition to increased expression of GATA-3 genes, the ratio of expression levels of GATA-3: Tbet genes in a biological isolate is higher than a given control value. In this case, as a control value, you can use the corresponding value of the ratio of the expression levels of GATA-3: Tbet genes in a healthy person isolate. Determining the ratio of GATA-3: Tbet gene expression levels increases the reliability of the judgment, since in addition to the expression of GATA-3 genes, Tbet gene expression is detected as an additional parameter. Since, as described in the introduction, the regulation of both transcription factors during their expression occurs in different directions, the inclusion of Tbet is an internal control tool for quantifying the expression of GATA-3 genes.

According to a preferred refinement, the method according to the invention for assigning a patient to the molecular phenotype of the “Th2-high” subgroup comprises the steps of:

- isolation of proteins or RNA from cells of a biological isolate of a patient;

- determination of the expression level of proteins or mRNA for GATA-3 and / or Tbet;

- inclusion of the patient in the “Th2-high” subgroup, if at least one of the conditions listed above under points a) or b) is fulfilled.

As an alternative, in addition to the indicated determination of the expression of GATA-3 and / or Tbet genes, other parameters are also determined for reliable inclusion in the “Th2-high” subgroup. So, for example, you can measure the level of IgE in serum and the number of eosinophilic granulocytes. Assignment to the “Th2-high” subgroup occurs if the serum IgE level is then higher than 100 IU / ml and / or if the number of eosinophilic granulocytes is 0.14 × 109 cells per liter or higher. Alternatively, if necessary, determination of the concentration of nitric oxide in exhaled air, i.e. determination of the value of FeNO.

Another preferred embodiment of the method according to the invention includes assigning a patient to one or another molecular phenotype of a “Th2-low” subgroup if at least one of the following conditions is true:

a) expression of GATA-3 genes in a biological isolate below a predetermined control value,

b) the ratio of the expression levels of GATA-3: Tbet genes in the biological isolate below a predetermined control value.

Thus, the “Th2-low” subgroup is characterized by low absolute expression of GATA-3 genes compared to a given control value. At the same time, as a control value - in the case of determining the amount of proteins - the corresponding value of the GATA-3 protein content in the isolate of a healthy person can be used. It should be borne in mind that the patient has a “Th2-low” subgroup with a disease accompanied by chronic inflammation, the absolute expression of the GATA-3 genes, however, will usually be higher than in the isolate of a healthy person. However, the absolute expression of GATA-3 genes may also be lower than in a healthy person. But, in any case, the expression of GATA-3 genes is not as high as described for the "Th2-high" subgroup. Thus, the patient is assigned to the “Th2-low” subgroup if he has a GATA-3 protein content compared to a healthy person isolate, if it is elevated, then moderately. However, you can use fixed control values. When determining the amount of mRNA, the corresponding value of the amount of GATA-3 mRNA in the isolate of a healthy person can be used as a control value, and the patient is assigned to the “Th2-low” subgroup when the amount of GATA-3 mRNA is less than in the corresponding sample of a healthy person or, in any case, increased slightly. However, again, you can use fixed control values.

The assignment of a patient to the indicated molecular phenotype of the “Th2-low” subgroup in accordance with a preferred embodiment of the method according to the invention occurs if, as an alternative or in addition to reduced expression of GATA-3 genes, the ratio of expression levels of GATA-3: Tbet genes in the biological isolate is lower than control value. In this case, the corresponding value of the ratio of the expression levels of GATA-3: Tbet genes in the isolate of a healthy person can be used as a control value. Determining the ratio of the expression levels of GATA-3: Tbet genes here also increases the reliability of the judgment, since in addition to the expression of GATA-3 genes, the expression of Tbet genes is established as an additional parameter.

Since, as described in the introduction, the regulation of both transcription factors in the process of their expression occurs in different directions, the inclusion of Tbet is to some extent an internal control tool for quantifying the expression of GATA-3 genes.

According to a preferred refinement, a method for assigning a patient to the molecular phenotype of a “Th2-low” subgroup according to the invention comprises the following steps:

- isolation of proteins or RNA from cells of a biological isolate of a patient;

- determination of the expression level of proteins or mRNA for GATA-3 and / or Tbet;

- inclusion of the patient in the “Th2-low” subgroup, if at least one of the conditions listed above under points a) or b) is fulfilled.

As an alternative, in addition to the indicated definition of expression of GATA-3 and / or Tbet genes, other parameters are again determined for reliable inclusion in the “Th2-low” subgroup. So, for example, you can measure the level of IgE in serum and the number of eosinophilic granulocytes. Assignment to the “Th2-low” subgroup occurs if the serum IgE level is lower than 100 IU / ml, and / or if the number of eosinophilic granulocytes is less than 0.14 × 10 ⁹ cells per liter. Alternatively, if necessary, the concentration of nitric oxide in exhaled air, i.e. The determination of the FeNO value has been performed.

According to another embodiment of the invention, a patient is assigned to one or another molecular phenotype of a “Th1-high” subgroup if at least one of the following conditions is fulfilled:

a) expression of Tbet genes in a biological isolate above a predetermined control value,

b) the ratio of the levels of expression of Tbet: GATA-3 genes in the biological isolate is higher than a predetermined control value.

Thus, the “Th1-high” subgroup is characterized by high absolute expression of Tbet genes compared to a given control value. Moreover, as a control value - in the case of quantitative determination of proteins - the corresponding value of the Tbet protein content in the isolate of a healthy person can be used, and the patient is assigned to the “Th1-high” subgroup if he has a high content of Tbet protein. However, you can also use fixed control values. In the case of determining the amount of mRNA as a control value, you can use the corresponding value of the amount of Tbet mRNA in the isolate of a healthy person, and the patient is assigned to the "Th1-high" subgroup if he has an increased amount of Tbet mRNA. However, again, you can use fixed control values.

The patient is assigned to the indicated Th1-high molecular phenotype of the subgroup in accordance with the preferred embodiment of the method according to the invention if, as an alternative or in addition to increased expression of Tbet genes, the ratio of Tbet: GATA-3 gene expression levels in the biological isolate is higher than the specified control value . Moreover, as a control value, you can use the corresponding value of the ratio of gene expression Tbet: GATA-3 in the isolate of a healthy person. Determination of the ratio of expression levels of Tbet: GATA-3 genes also increases the reliability of the judgment, since in addition to the expression of Tbet genes, the expression of GATA-3 genes is established as an additional parameter, and the inclusion of GATA-3 is an internal control tool for the quantitative determination of Tbet gene expression.

According to a preferred refinement of the method for assigning a patient to the molecular phenotype of a “Th1-high” subgroup according to the invention, the following steps are included:

- isolation of proteins or RNA from cells of a biological isolate of a patient;

- determination of the expression level of proteins or mRNA for Tbet and / or GATA-3;

- inclusion of the patient in the "Th1-high" subgroup, if at least one of the above conditions under points a) or b) is fulfilled.

Another aspect of the present invention relates to a method that allows the patient to be assigned to one or another molecular phenotype of the "Th1-low" subgroup, if at least one of the following conditions is met:

a) expression of Tbet genes in a biological isolate below a predetermined control value,

b) the ratio of the expression levels of Tbet GATA-3 genes in the biological isolate below a predetermined control value.

Thus, the “Th1-low” subgroup is characterized by low absolute expression of Tbet genes compared to a given control value. In this case, as a control value - in the case of quantitative determination of proteins - you can use the corresponding value of the protein content Tbet in the isolate of a healthy person. It should be borne in mind that in a patient of the “Th1-low” subgroup with a disease accompanied by chronic inflammation, the absolute expression of Tbet genes, however, may be higher than in the isolate of a healthy person. However, the absolute expression of Tbet genes may also be lower than in a healthy person. But, in any case, the expression of Tbet genes is not as high as described for the "T1h-high" subgroup. Thus, the patient is assigned to the “Th1-low” subgroup if he has a Tbet protein content compared to a healthy person isolate, if it is elevated, then moderately. However, you can use fixed control values. In the case of determining the amount of mRNA, the corresponding value of the amount of Tbet mRNA in the isolate of a healthy person can be used as a control value, and the patient is assigned to the “Th1-low” subgroup when the quantity of Tbet mRNA is lower than in the corresponding sample of a healthy person or, in in any case, increased slightly. However, again, you can use fixed control values.

The assignment of a patient to the indicated molecular phenotype of the “Th1-low” subgroup in accordance with a preferred embodiment of the method according to the invention occurs if, as an alternative or in addition to reduced expression of Tbet genes, the ratio of Tbet: GATA-3 gene expression levels in the biological isolate is below a predetermined control value . Moreover, as a control value, the corresponding value of the ratio of the levels of expression of Tbet: GATA-3 genes in the isolate of a healthy person can be used. The determination of the ratio of Tbet: GATA-3 gene expression levels here also increases the reliability of the judgment, since in addition to the expression of Tbet genes, the expression of GATA-3 genes is established as an additional parameter, and the inclusion of GATA-3 to some extent represents an internal control tool for the quantitative determination of Tbet gene expression.

According to a preferred refinement, the method of assigning a patient to the molecular phenotype of the “Th1-low” subgroup according to the invention comprises several steps:

- isolation of proteins or RNA from cells of a biological isolate of a patient;

- determination of the expression level of proteins or mRNA for Tbet and / or GATA-3;

- inclusion of the patient in the “Th1-low” subgroup, if at least one of the conditions listed above under points a) or b) is fulfilled.

As an alternative, in addition to the indicated definition of GATA-3 and / or Tbet gene expression, other parameters can be determined for reliable inclusion in the “Th1-high” and “Th1-low” subgroup. So, for example, you can determine the number of eosinophilic granulocytes or quantitatively determine the level of IgE in serum. Assignment to the “Th1-high” subgroup occurs if the serum IgE level is lower than 100 IU / ml, and / or if the number of eosinophilic granulocytes is less than 0.14 × 10 ⁹ cells per liter. Otherwise, the patient is assigned to the “Th1-low” subgroup. Alternatively, if necessary, you can also determine the concentration of nitric oxide in exhaled air, i.e. determine the value of FeNO.

To account for differences in sample preparation, normalization of GATA-3 and Tbet concentrations can be performed. Differences in sample preparation can occur, for example, due to different numbers of cells subjected to lysis, as a result of different levels of lysis efficiency in individual samples, or due to different contents of different types of cells in cell preparations. According to the invention, among other things, the following standardization options exist: standardization according to the total protein content in the cell lysate, standardization according to the number of lysed cells, or standardization according to the concentration of specific marker proteins that are found specifically in certain types of cells.

Patients with a diagnosed molecular phenotype of a “Th2-high” subgroup under certain circumstances can simultaneously be placed in a “Th1-low" subgroup. Also, patients with a diagnosed molecular phenotype of the “Th1-high” subgroup can simultaneously be assigned to the “Th2-low” subgroup under certain circumstances. This is due to the fact described above that polarization in Th2 inhibits the differentiation of Th1– by suppressing Tbet and vice versa.

In the framework of the present invention, the diagnosis and treatment of diseases accompanied by chronic inflammation, for example, autoimmune diseases, diseases of the rheumatic clinical and morphological groups (manifestations, for example, on the skin, lungs, kidneys, circulatory system, nervous system, connective tissue, supporting motor apparatus, endocrine system), allergic reactions of the immediate type and asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, psoriasis and contact eczema, as well as chronic rejection reactions after organ transplantation and bone marrow. Diagnosis and / or treatment of neoplastic diseases is also possible according to the invention if GATA-3 or Tbet are involved in the occurrence and / or if, as a secondary phenomenon, their regulatory action is terminated.

In the framework of the present invention, a chronic inflammatory disease is caused either by Th2, for example, allergic bronchial asthma, rhinoconjuctivitis, allergic sinusitis, atopic dermatitis, food allergies, pemphigus, ulcerative colitis, parasitic diseases, or Th1, for example, psoriasis, allergic contact eczema, regional enteritis, COPD, rheumatoid arthritis, autoimmune diseases, type 1 diabetes mellitus or multiple sclerosis.

Further, according to the invention, the aforementioned problem is solved by a medicament for treating diseases accompanied by chronic inflammation in a patient with a particular molecular phenotype, the molecular phenotype being determined using a method in accordance with one or more embodiments of the diagnostic method according to the invention. Moreover, the identified molecular phenotype includes, in particular, the groups “Th1-low”, “Th1-high”, “Th2-low” or “Th2-high”.

According to a preferred embodiment, said medicament comprises a GATA-3 or Tbet-specific ribonucleic acid or deoxyribonucleic acid, in particular a GATA-3 or Tbet-specific DNA enzyme.

The general model of the DNA enzyme is the 10-23 model (Sontoro et al., 1997). DNA enzymes of model 10-23, also referred to as “10-23 DNA enzymes", contain a catalytic domain of 15 nucleotides with two substrate-binding domains on each side. Moreover, the catalytic domain preferably contains the sequence ggctagctacaacga (SEQ ID No. 154). The length of the substrate-binding domains can vary, the domains can have either the same or different lengths. In one preferred embodiment, the length of the substrate-binding domains is from 6 to 14 nucleotides, it is particularly preferred that in each case there are at least nine nucleotides. Such DNA enzymes contain the general sequence nnnnnnnnnggctagctacaacgannnnnnnnnn (SEQ ID NO 155). Particularly preferred are the substrate binding domains that bind mRNA, with coding for the GATA-3 and Tbet proteins.

Said ggctagctacaacga catalytic central domain is only one preferred embodiment. One skilled in the art knows that “10-23 DNA enzymes” with comparable biological activity can be stored with a modified catalytic domain.

In one particularly preferred embodiment, the substrate-binding domains are completely complementary to the site that is adjacent to the cleavage site. However, for the binding and cleavage of the target RNA, the DNA enzyme does not have to be fully complementary. Type 10-23 DNA enzyme cleaves the target mRNA into sequential purine-pyrimidine chains. In the framework of the present invention, DNA enzymes preferably contain in vivo active anti-GATA-3 and Tbet DNA enzymes according to WO 2005/033314 A2, the contents of which are set forth in relation to the scope of the disclosure of the present invention.

A medicament for specifically inhibiting GATA-3 expression in vivo contains, in particular, at least one DNA enzyme selected from the group consisting of DNA enzymes with one sequence according to one of the sequences from SEQ ID NO 1 to SEQ ID NO 70. Such a DNA enzyme binds preferably to an mRNA encoding for a human GATA-3 gene with a nucleotide sequence of a gene selected from SEQ ID NO 151 (human GATA-3 from database No. XM 043124), SEQ ID NO 152 ( GATA-3 people from the database No: X58072) and SEQ ID NO 153 (GATA-3 people a, sequenced from plasmid pCR2.1).

A drug for specific inhibition of GATA-3 expression in vivo preferably contains the hgd40 DNA enzyme with the sequence 5'-GTGGATGGAggctagctacaacgaGTCTTGGAG (SEQ ID NO 40).

A drug for specific inhibition of Tbet expression in vivo contains in particular at least one DNA enzyme selected from a series of DNA enzymes with a sequence in accordance with one of the sequences from SEQ ID NO 71 to SEQ ID NO 148. Such DNA the enzyme binds preferably to mRNA that encodes for the human Tbet gene with a nucleotide sequence of a gene selected from the sequences SEQ ID NO 149 (Tbet person from database no: NM 013351) and SEQ ID NO 150 (human Tbet sequenced from pBluescript-SK) .

A drug for specific inhibition of Tbet expression in vivo preferably contains the td69 DNA enzyme with the sequence 5'-GGCAATGAAggctagctacaacgaTGGGTTTCT (SEQ ID NO 139) or td70 with the sequence 5'-TCACGGCAAggctagctacaacgaGAACTGGGT (SEQ ID NO 140).

As an alternative to DNA enzymes, a drug for specifically inhibiting the expression of GATA-3 or Tbet may contain a suitable small interfering ribonucleic acid.

The drug preferably has a composition that is able to administer to patients the indicated specific molecules of ribonucleic or deoxyribonucleic acid in the form of a pharmaceutically acceptable composition, either orally, rectally, parenterally, intravenously, intramuscularly, or subcutaneously, intracisternally, intravaginally, intraperitoneally, intrathecally, intravascularly, locally ( ointment or drops) or in the form of an aerosol. Dosage forms for topical administration of the drug according to this invention include ointments, powders, aerosols or inhalants. The sterile active ingredient is mixed with a physiologically acceptable excipient and, depending on the needs, with possible preservatives, buffers or blowing agents.

The drug can be used to treat all groups of diseases accompanied by chronic inflammation.

According to a particularly preferred embodiment of the present invention, the treatment of patients with a Th2-high subgroup molecular phenotype is carried out using a GATA-3 specific DNA enzyme. Therapy of a patient with the molecular phenotype of the “Th1-high” subgroup is carried out using Tbet-specific DNA enzyme. Further, the treatment of a patient with the molecular phenotype of the “Th2-low” subgroup is carried out using a Tbet-specific DNA enzyme, and the treatment of the patient with the molecular phenotype of the “Th2-low” subgroup can be performed using the GATA-3 DNA enzyme.

Thus, a drug according to the invention with a GATA-3-specific DNA enzyme is preferably provided for treating a patient with a Th2-high subgroup molecular phenotype, and a drug with a Tbet-specific DNA enzyme is preferably provided for treating a patient with a molecular phenotype " Th1-high subgroups.

According to the invention, a particular advantage of the medicament according to one of the above embodiments for the specific treatment of a patient subgroup diagnosed according to the invention — namely, “Th2-high”, “Th2-low,“ Th1-high ”or“ Th1-low ”- is the fact that with the help of a specific drug, in particular, a DNA enzyme and / or a small interfering RNA, there is a functional inactivation of just those coding RNA molecules of transcription factors that previously had differential expression has been updated, and which are involved in the occurrence of chronic inflammatory reactions and autoimmune diseases. This strategy is clearly different from traditional approaches, as well as from the mentioned procedure described in the document by Corren et al., 2011, since there, on the one hand, the amount of periostin (surrogate marker) was determined, but then they offered therapy against another target, for example, against IL-13 using an anti-IL-13 antibody. The drug according to the invention, in contrast, is highly specific. It causes cell-specific interference and is specific to departments and organs.

Formulations for the manufacture of a medicament according to the invention include pharmaceutically acceptable compositions containing modifications and “prodrug forms” (inactive forms of the drug) if, according to a reliable medical assessment, they do not cause excessive toxicity, irritation or allergic reactions in the patient. The term "prodrugs" refers to compounds that are transformed, for example, by hydrolysis in the blood, to improve the input into the body.

The drug according to the invention can also be used in the form of a heterogeneous emulsion for the application of these specific nucleic acid molecules. To this end, said heterogeneous emulsion comprises an external aqueous phase W1, an oil phase 0 dispersed in an external aqueous phase W1, and an internal aqueous phase W2 dispersed in an oil phase 0, wherein at least one alkaline electrolyte is selected in an internal aqueous phase W2 and alkaline earth halides and sulfates, and at least one specific molecule of ribonucleic acid or deoxyribonucleic acid, preferably GATA-3 or Tbet-specific DNA enzyme, wherein the external aqueous phase W1 contains um hydrophilic emulsifier, which is a polymer of ethylene oxide and propylene oxide, and the oil phase is formed triacylglycerides 0 and contains a number of lipophilic emulsifier dimethicones. Using one such heterogeneous emulsion, it is possible, in particular, to protect nucleic acid molecules from undesirable destruction in particular.

The type of dosage is determined by the attending physician taking into account specific clinical factors. The specialist knows that the type of dosage depends on various factors, for example, on the body size, weight, skin condition, age, gender or general health of the patient, but also on the particular medication taken, duration and method of administration, as well as other medications which can be given in parallel. Moreover, in accordance with a particularly preferred embodiment, the amount of the active component of the drug can be adapted taking into account the measured level of expression. So, as a result of inclusion in the “Th2-high” subgroup and with very high expression of GATA-3 genes detected, an increased dose of the active component, in particular, a GATA-3-specific enzyme, can be given. Accordingly, after inclusion in the “Th1-high” subgroup and when very high expression of Tbet genes is detected, an increased dose of the active component, in particular Tbet-specific DNA enzyme, can be given.

Another aspect of the present invention relates to a kit for diagnosing the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation, the kit containing at least one specific component for the quantification of GATA-3 and / or Tbet protein or mRNA in a patient's biological isolate.

The diagnostic reagent kit according to the invention can easily be offered as a ready-to-use “kit” comprising an antibody or antigen adsorbed on the surface of a carrier and a human IgG antibody composition, which, for example, in the case of humans, is an IgG antibody composition for human cells, labeled in such a way that they are detected through a cascade of reactions such as biotin-streptavidin-peroxidase or alkaline phosphatase.

Alternatively, the diagnostic kit along with the carrier also contains a buffer and reagents, for example, reagents necessary to confirm the reaction, for example, streptavidin, which is associated with a marker that gives a staining reaction.

Alternatively, the kit further comprises a GATA-3 and / or T-Bet sample standard for calibrating the kit, and one sample standard is used to detect the amount of protein or mRNA in GATA-3 and / or Tbet.

In a preferred embodiment, a GATA-3 and / or Tbet-specific antibody is contained in each case to determine the amount of protein. If necessary, according to one of the options, other components may be contained for carrying out any method of immunological analysis, in particular, enzyme-linked immunosorbent assay (ELISA).

Another component for carrying out an enzyme-linked immunosorbent assay (ELISA) is selected from the following: lysis buffer for cell lysis, microtiter plate, quantitative protein standards for GATA-3 and / or Tbet, secondary antibodies and an associated enzyme for working with a substrate for detection. Along with the first GATA-3 and / or Tbet specific antibody, it is preferable to use another GATA-3 or Tbet specific antibody from the kit.

For quantitative determination of mRNA, the kit may contain in each case a site-specific probe and / or primer for the GATA-3 and / or Tbet genes.

Other features, details and advantages of the invention follow from the claims, as well as from the following description of examples of embodiments of the invention using the drawings.

A brief description of the graphic materials

FIG. 1 Effect of various detergents on the release of GATA-3 from stimulated Jurkat cells,

FIG. 2a, 2b The results of the quantitative determination of Tbet and GATA-3 using the "sandwich" variant of enzyme-linked immunosorbent assay using a chromogenic substrate (ELISA),

FIG. 3 Calibration curve of enzyme-linked immunosorbent assay (ELISA) GATA-3 for the quantification of samples

FIG. 4 Calibration curve of the solid-state enzyme-linked immunosorbent assay (ELISA) Tbet for the quantification of samples,

FIG. 5 Normalized determination of Tbet in lysates of mononuclear cells of human peripheral blood (PBMC) in healthy subjects and allergy sufferers,

FIG. 6 Significant improvement in allergic airway inflammation after four days of treatment with the GATA-3-specific hgd40 DNA enzyme (SEQ ID NO 40) compared to untreated mice,

FIG. 7 The effect of GATA-3-specific hgd40 DNA enzyme (SEQ ID NO 40) on the number of neutrophils resulting from chronic inflammation, the number of eosinophils in BAL and the isolation of IL-5 (interlekin-5) after eight weeks of treatment, and

FIG. 8 Effect of GATA-3-specific hgd40 DNA enzyme (SEQ ID NO 40) on peribronchial / perivascular inflammation and goblet cell hyperplasia in lung tissues.

Materials and methods:

Cells can be isolated, for example, using technologies based on the binding of specific antibodies. Magnetic microcarrier pellets supplied by Miltenyi (Macs system), Dynal (DynaBeads microcarrier granules) or BD-Bioscience (iMAG) are used here. Alternatively, this is achieved by purifying the cells with a fluorescently-labeled antibody in cell sorters, for example, manufactured by Cytomation (MOFLO) or BD-Bioscience (FACS-Vantage). The purity of the target cells is preferably at least 80%, more preferably at least 95%, and most preferably at least 99%.

RNA isolation methods are described, for example, by Sambrook and Russell, Molecular Cloning (A Cloning of a Nucleic Acid Molecule), A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory (2001), New York, and Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons (1998), New York. In addition, to isolate RNA, one of ordinary skill in the art can use commercially available kits (silica technology), for example, the Qiagen RNeasy kit. It is further preferred to isolate and purify mRNA from target cells by using commercially available kits, for example, manufactured by Qiagen (Oligotex mRNA Kit), Promega (mRNA PolyATract Isolation System mRNA isolation system), or Miltenyi (mRNAdirect).

Examples of carrying out the invention

An example embodiment of the invention 1

GATA-3 and Tbet are proteins that act as so-called transcription factors in the nucleus of T-helper cells of the Th1 and Th2 subtype. To determine the concentration of both of these nuclear proteins in a certain volume of biological isolate, in particular, in a certain volume of whole blood, the cells forming GATA-3 and Tbet must first be isolated and then lysed. Direct detection of these proteins from human serum or plasma is not possible, because they are contained there in an undetectable concentration. Therefore, the analysis of GATA-3 and Tbet is performed in 4 stages:

- separation and isolation of cells expressing GATA-3 / Tbet from other cellular components of whole blood;

- lysis of cells and isolation of intracellular / nuclear proteins;

- quantitative determination of the concentration of GATA-3 and Tbet;

- rationing of detected concentrations of GATA-3 and Tbet

Separation and isolation of cells expressing GATA-3 / Tbet from other cellular components of whole blood

For this, methods of varying complexity are used, in particular for the separation and isolation in the framework of the present invention, the following steps are performed:

- isolation of leukocytes from whole blood by centrifugation in a density gradient of ficoll followed by affinity purification of Th1 / Th2 cell types using antibodies to specific cell surface markers,

- if necessary, affinity purification of Th1 / Th2 cell types using antibodies to specific cell surface markers can also be performed as a 1-step method without first increasing the concentration of leukocytes; in certain situations, to quantify the GATA-3 and Tbet proteins, it is sufficient to isolate the white blood cells by centrifugation in a density gradient of ficoll from whole blood;

- if necessary, instead of centrifuging in a density gradient of ficoll, it is also possible to use based on the study of chains of granules of affinity purification of Th1 / Th2 cell types using antibodies to specific cell surface markers in a 96-well deep-well plate;

- if necessary, instead of centrifuging in a density gradient of ficoll, it is also possible to use affinity purification of leukocytes based on the study of granule chains using antibodies to specific cell surface markers, for example, in a 96-well deep-well plate;

- if necessary, to obtain a leukocyte preparation, hypoosmotic lysis of red blood cells can be performed; or

- if necessary, the lysis of proteins can be carried out immediately from whole blood.

Cell Lysis and Intracellular / Nuclear Protein Isolation

For this, various methods and principles are applied, in particular, in the framework of the present invention, the following process steps are performed:

- destruction of cell membranes with lysis buffer based on the following principles of action:

a) hypotonic lysis buffers, under the influence of which the cells burst;

b) detergent-containing buffers that destroy the cell membrane and thus release intracellular proteins

c) buffers with high ionic strength and / or osmotically active buffers that dehydrate cells and thereby violate cellular integrity.

- physical methods, for example, heating, shock freezing or ultrasound

- mechanical methods, for example, homogenization or grinding in a mortar.

Possible examples of detergent buffers include:

- buffer systems with a high concentration of ionic (e.g. SDS or Cholat and their derivatives) or non-ionic (e.g. Triton or Tween-20) detergents

- mixtures of ionic and nonionic detergents (for example, Ripa buffer with 50 mM Tris ⋅ HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS sodium dodecyl sulfate)

- commercially available lysis buffers of unknown composition (e.g. M-PER)

The effect of different detergents on the release of GATA-3 stimulated Jurkat cells is shown in FIG. 1. In this case, lysis of Jurkat cells (human T-cell line) through various lysis buffers and quantification of GATA-3 using enzyme-linked immunosorbent assay (ELISA) found a particularly abundant isolation of GATA-3 protein using RIPA buffer (1% RIPA). It was possible to identify approximately 50 ng / ml GATA-3.

Quantification of the concentration of GATA-3 and Tbet

The concentration of both transcription factors GATA-3 and Tbet can be determined in fundamentally different ways. In the framework of the present invention to them, in particular, include:

enzyme-linked immunosorbent assay (ELISA) enzyme-linked immunosorbent assay with chemiluminescence (CLIA)

- solid phase immunoassay with fluorescence amplification (FLIA)

- mass spectrometric methods

- chromatographic methods (e.g. gas chromatography)

- liquid methods with separation of the solid phase (for example, high performance liquid chromatography, HPLC)

- micro and nanofluidic technologies

In FIG. 2a and FIG. Figure 2b shows the results of the quantitative determination of Tbet and GATA-3 by the "sandwich" variant of enzyme-linked immunosorbent assay using a chromogenic substrate (ELISA). Cells were obtained by centrifugation in a density gradient ficoll from whole blood. Cells (stimulated human mononuclear cells) were lysed with Ripa buffer. The lysate was examined for concentrations of GATA-3 and Tbet using two prototype enzyme-linked immunosorbent assays (ELISA). The concentration of both proteins is depicted relative to the total protein concentration in cell lysates (normalization for protein content).

The results of FIG. 2a say that Th1 cells show a higher Tbet content (approx. 160 ng / ml of analyte / mg protein) than Th2 cells (approx. 56 ng / ml of analyte / mg protein), and that this clearly follows from the results of the analysis of ELISA (enzyme-linked immunosorbent assay):

According to FIG. 2b, the content of GATA-3 in Th2 cells (approximately 10 ng / ml of analyte / mg protein) is higher compared to the content in Th1 cells (approximately 6 ng / ml of analyte / mg protein). Thus, the content of GATA-3- in Th2 cells is higher by more than 1.5 times, and more precisely, by about 1.7 times, than in Th1 cells.

In addition, from FIG. 2a and 2b, it can be understood that when normalizing for protein content, the quantitative ratio Tbet: GATA-3 in Th1 cells differs significantly from the corresponding ratio in Th2 cells. Thus, the quantitative ratio Tbet: GATA-3 here in Th1 cells is approximately 27, i.e. greater than 20, while the corresponding quantitative ratio in Th2 cells is approximately 6, i.e. less than 10.

The quantitative determination of GATA-3 and Tbet in each case is carried out using the “sandwich” variant of enzyme-linked immunosorbent assay (ELISA).

An example embodiment of the invention 2 - GATA-3 enzyme-linked immunosorbent assay (ELISA):

For this, 96-well microtiter plate wells are coated with GATA-3 specific antibodies. After adding the sample and / or reference, GATA-3 forms a binding to antibodies on a 96-well plate. After washing, a second, specific biotinylated anti-GATA-3 antibody is added to remove unbound substances. After another wash, peroxidase-labeled streptavidin is added to remove unbound substances. After washing, a substrate is added to remove unbound substances. Color development ends after a predetermined period of time by adding a stop reagent. Quantify the intensity of color development using a photometric microplate reader (reader). Quantitative determination of samples is performed by comparison with existing standards with known protein concentration. In FIG. Figure 3 shows the corresponding calibration curve of the enzyme-linked immunosorbent assay (ELISA) GATA-3.

According to an example of an embodiment of the invention, in order to carry out the GATA-3 enzyme-linked immunosorbent assay (ELISA), in particular, the following steps are performed:

- placement of the required number of holes in the frame of a 96-well plate

- adding a buffer solution for the study in the amount of 50 μl / well

- add 100 μl / well of reference / control / sample incubation for 60 minutes in a shaker

- 4-fold washing of all wells with washing buffer each time in an amount of 400 μl / well

- adding biotinylated anti-GATA-3 antibodies in a volume of 100 μl / well

- incubation for 60 minutes in a shaker

- 4-fold washing of all wells with washing buffer in an amount of 400 μl / well

- adding labeled peroxidase streptavidin in an amount of 100 μl / well

- incubation for 30 minutes in a shaker

- 4-fold washing of all wells with washing buffer in an amount of 400 μl / well

- adding substrate in an amount of 100 μl / well incubation for 30 minutes

- termination of the reaction by adding 100 μl of a stop reagent quantitative determination of optical density at 450 nm using a photometric microplate reader (reader)

An example embodiment of the invention 3 - enzyme-linked immunosorbent assay (ELISA) Tbet:

The detection of Tbet protein is carried out in accordance with the following research principle: Tbet is quantitated using a “sandwich” variant of enzyme-linked immunosorbent assay (ELISA). For this, a layer of antibodies specific for Tbet is applied to the wells of a microtiter plate in 96 wells. After adding a sample and / or reference, Tbet forms a binding to antibodies on a 96-well plate. After washing, a second Tbet specific antibody is added to remove unbound substances. After another wash to remove unbound substances, a peroxidase-labeled antibody specific for Tbet-specific antibody is added. After the last wash, a substrate is added to remove unbound substances. Color development ends after a predetermined period of time by adding a stop reagent. Quantitative determination of the intensity of color manifestations is performed using a photometric microplate reader (reader). Quantitative determination of samples is carried out by comparison with existing standards with known protein concentration. Figure 4 shows the corresponding calibration curve of enzyme-linked immunosorbent assay (ELISA) Tbet.

In accordance with an example of an embodiment of the invention, the following steps are used in particular for the Tbet Enzyme Immunoassay (ELISA):

- placement of the required number of holes in the frame of a 96-well plate

- adding a buffer solution for the study in the amount of 50 μl / well

- adding 100 μl / well of reference / control / sample

- incubation for 60 minutes in a shaker

- 4-fold washing of all wells with washing buffer each time in an amount of 400 μl / well

- adding anti-Tbet antibodies in a volume of 100 μl / well

- incubation for 60 minutes in a shaker

- 4-fold washing of all wells with washing buffer in an amount of 400 μl / well

- the addition of peroxidase-labeled anti-Tbet-specific antibodies in a volume of 100 μl / well

- incubation for 30 minutes in a shaker

- 4-fold washing of all wells with washing buffer in an amount of 400 μl / well

- adding substrate in an amount of 100 μl / well

- incubation for 30 minutes

- termination of the reaction by adding 100 μl of stop reagent

- quantitative determination of optical density at 450 nm s

using a photometric microplate reader (reader)

Rationing of concentrations of GATA-3 and Tbet

To account for differences in sample preparation, standardization of GATA-3 and Tbet concentrations can be performed. Differences in sample preparation may occur, for example, for the following reasons:

- different number of cells subjected to lysis

- different lysis efficiencies in individual samples, or

- different contents of various cellular types of cell preparations.

According to the invention, among other things, the following standardization options exist:

- rationing on the total protein content in the cell lysate (see paragraph "Quantitative determination of the concentration of GATA-3 and Tbet")

- rationing on the number of cells that has been lysed (see Fig. 5), or

- rationing on the concentration of specific marker proteins found specifically in certain cell types.

In FIG. 5. The normalized determination of Tbet in lysates of human peripheral blood mononuclear cells (PBMCs) is presented. In this case, mononuclear cells of peripheral blood of healthy subjects, as well as patients suffering from allergic diseases, for example, allergic bronchial asthma, rhinoconjunctivitis, allergic sinusitis, atopic dermatitis, and food allergies, were lysed. Tbet concentration was normalized by the number of cells in lysates. The disease depends on Th2 cells, and it is logical that allergy sufferers were able to detect a lower concentration of Tbet (about 12 ng / ml / 1 million cells) compared with healthy subjects (about 27 ng / ml / 1 million cells). Thus, the concentration of Tbet in an allergic person was slightly more than 2 times lower than in healthy subjects. Therefore, patients can easily be attributed to the “Th1-low” molecular phenotype, because expression of Tbet genes in a biological isolate below a predetermined control value; here, expression of Tbet genes of healthy subjects.

An example embodiment of the invention 4

In the modification of Examples 2 and 3 in accordance with Example 4 for preparing samples, Th1 / Th2 cells are enriched with magnetic microcarrier beads that are coated with cell-specific antibodies.

Then, GATA-3 is detected according to the procedure described in Example 2.

An example embodiment of the invention 5

In the modification of examples 2 and 3 in accordance with example 5 for the preparation of samples, white blood cells are enriched by gel filtration. Then make the identification of GATA-3 in the order described in example 2.

An example embodiment of the invention 6

The GATA-3-specific DNA enzyme demonstrates therapeutic effects in a mouse model of an ovalbumin-induced allergic airway inflammation of the “Th2-high” phenotype.

In order to best reproduce the clinical “Th2-high” phenotype in a mouse model, BALB / c mice were sensitized with the model allergen ovalbumin (OVA) in the presence of adjuvant Al (OH) ₃ on days 0, 14 and 21 by intraperitoneal injections. On days 24-26, the mice inhaled ovalbumin in the form of a 1% aerosol to cause an allergic inflammatory reaction in the lung with Th2 dominance. On the 23-26th day, intranasal applications were made of the GATA-3 specific DNA enzyme hgd40 (SEQ ID NO 40) dissolved in phosphate-buffered saline.

Moreover, the Balb / c mouse line is characterized in that it preferably generates powerful Th2 responses. This effect is enhanced by the use of AL (OH) ₃ as an adjuvant, significantly supporting the formation of immune responses with Th2 dominance. The described mouse model is suitably characterized by massive infiltration of eosinophilic and Th2 cells in the airways, accompanied by hyperplasia of mucus-forming goblet cells with enhanced mucus production, as well as the formation of airway hyperreactivity. From an immunological point of view, along with allergen-specific Th2 cells, also characterized by the production of typical cytokines IL-4, IL-5 and IL-13, ovalbumin-specific antibodies of the immunoglobulin classes IgE and lgG1 can also be established (in a mouse both Th2-dependent ) All of these parameters are typical clinical features of the “Th2-high” phenotype (Wenzel et al., Am J Respir Crit Care Med. 1999 Sep; 160 (3): 1001-8; Woodruff et al., 2009). At the same time, the reaction force in some parameters in the experimental model on the animal is even more pronounced than in the clinical situation of the patient; for example, eosinophilic granulocytes account for approximately 60-70% of all leukocytes in bronchoalveolar washout (BAL) in the mouse model, while already 3-5% of these cells in the patient’s sputum indicate a phenotype with Th2 dominance.

In accordance with FIG. 6, after a four-day treatment with hgd40-specific GATA-DNA enzyme (SEQ ID NO 40), a significant improvement in allergic airway inflammation was found compared to untreated mice. First of all, the number of eosinophils in the bronchoalveolar washout (BAL) was reduced. In addition, it was possible to significantly reduce the concentrations of the characteristic “Th2-high” phenotype of the cytokines IL-5 and IL-13 in BAL.

An example embodiment of the invention 7

GATA-3-specific DNA enzyme exhibits significant therapeutic effects in a mouse model of chronic allergic airway inflammation with Th2 dominance.

To best reproduce the clinical “Th2-high” phenotype in the mouse model of chronic disease, BALB / c mice were sensitized with ovalbumin (OVA) in the presence of adjuvant Al (OH) ₃ on days 0, 14 and 21 by intraperitoneal injection . By conducting provocation tests twice a week with ovalbumin in the form of an aerosol for 14 weeks, chronic airway inflammation was caused in mice. Over the past eight weeks, the therapy was carried out three times a week by intranasal applications with budesonide or the hdg40-specific DNA enzyme hdg40 (up to 121 days).

In accordance with FIG. 7 and 8, after eight weeks of treatment with the GATA-3-specific hgd40 DNA enzyme (SEQ ID NO 40), it was possible to significantly reduce the number of eosinophils in the BAL and also to note a decrease in the number of neutrophils resulting from chronic inflammation. At the same time, a decrease in peribronchial / perivascular inflammation and a decrease in goblet cell hyperplasia were found in lung tissues. At the same time, in the lymphocytes activated by the stimulator, in individuals treated with hgd40, a decreased release of IL-5 was observed. In the group in which budesonide was used, in contrast, no significant improvement was found.

The invention is not limited to the embodiments provided herein, and may be implemented in various modifications.

All of the following features and advantages from the claims, descriptions and figures of the drawings, including structural details, configurations in space and stages of the implementation of the methods, can be essential for the invention, both on their own and in different combinations.

Claims (21)

1. A method for diagnosing the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation, according to which the expression of the protein and / or mRNA GATA-3 and / or Tbet is quantified in the biological isolate of the patient, which is used to refer to one or another molecular phenotype of the disease, selected from the series that includes the subgroups "Th2-high", "Th2-low", "Th1-high" and "Th1-low", characterized in that - the patient is assigned to one or another molecular phenotype of the “Th2-high” subgroup if at least one of the following conditions is fulfilled: a) expression of GATA-3 in the biological isolate above a predetermined control value, b) a ratio of expression levels of GATA-3: Tbet in the biological isolate above a predetermined control value; - the patient is assigned to one or another molecular phenotype of the “Th2-low” subgroup if at least one of the following conditions is fulfilled: a) expression of GATA-3 in a biological isolate below a predetermined control value, b) the ratio of expression levels of GATA-3: Tbet in the biological isolate below a predetermined control value; - the patient is assigned to one or another molecular phenotype of the “Th1-high” subgroup if at least one of the following conditions is fulfilled: a) expression of Tbet in a biological isolate above a predetermined control value, b) a ratio of expression levels of Tbet: GATA-3 in the biological isolate above a predetermined control value; - the patient is assigned to one or another molecular phenotype of the "Th1-low" subgroup if at least one of the following conditions is fulfilled: a) expression of Tbet in a biological isolate below a predetermined control value, b) a ratio of expression levels of Tbet: GATA-3 in the biological isolate below a predetermined control value. 2. The method according to p. 1, characterized in that the expression of the protein is quantified by immunoassay, preferably enzyme-linked immunosorbent assay ELISA, radioimmunoassay, electrochemiluminescent immunoassay, enzyme-linked immunosorbent assay with CLIA, solid-state immuno-immunoassay immunoassay immunoassay immunoassay immunoassay; and / or mRNA expression is quantified, in particular, by PCR or microarray analysis. 3. The method according to p. 1 or p. 2, characterized in that along with the determination of the expression of the protein and / or mRNA GATA-3 and / or Tbet determine the level of IgE in the blood serum and / or the number of eosinophilic granulocytes and / or find the value of FeNo . 4. The method according to p. 1 or p. 2, characterized in that the disease accompanied by chronic inflammation is caused by Th2, in particular allergic bronchial asthma, rhinoconjunctivitis, allergic sinusitis, atopic dermatitis, food allergies, pemphigus, ulcerative colitis, parasitic diseases, or due to Th1, in particular psoriasis, allergic contact eczema, regional enteritis, COPD, rheumatoid arthritis, autoimmune diseases, type 1 diabetes mellitus or multiple sclerosis. 5. A medicament for treating a patient suffering from a disease accompanied by chronic inflammation, the patient having a molecular phenotype belonging to a number of subgroups “Th2-high”, “Th2-low”, “Th1-high” and “Th1-low”, defined according to the method according to one of paragraphs. 1-4, characterized in that it contains a specific GATA-3 or Tbet DNA enzyme, and the fact that the drug comprises a heterogeneous emulsion containing an external aqueous phase W1, dispersed in an external aqueous phase W1, an oil phase O and dispersed in an oil phase O the internal aqueous phase W2, wherein in the internal aqueous phase W2 at least one electrolyte selected from a series of alkaline and alkaline-earth halides and sulfates is provided, and at least one GATA-3 or Tbet-specific DNA enzyme, wherein the external aqueous phase is W1 contains gi a diphilic emulsifier, which is a polymer of ethylene oxide and propylene oxide, and the oil phase O is formed by triacylglycerides and contains a lipophilic emulsifier from a number of dimethicones. 6. The drug of claim 5, wherein the GATA-3-specific DNA enzyme is provided for treating a patient with a Th2-high subgroup molecular phenotype, and that the Tbet-specific DNA enzyme is provided for treating a patient with a molecular phenotype Th1-high subgroups. 7. A kit for diagnosing the molecular phenotype of a patient suffering from a disease accompanied by chronic inflammation, the kit containing at least one specific component for quantifying the expression of GATA-3 and / or Tbet protein and / or mRNA in a patient’s biological isolate according to the method of one of p. 1-4. 8. The kit according to p. 7, characterized in that for the quantitative determination of protein expression in each case contains an antibody specific for GATA-3 and / or Tbet, and preferably contains components for immunoassay, in particular enzyme-linked immunosorbent assay ELISA. 9. The kit according to p. 7, characterized in that for the quantitative determination of mRNA expression contains a site-specific probe and / or site-specific primer for the GATA-3 and / or Tbet genes.

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